EP1616005B1

EP1616005B1 - Mannosyl(1-2)glycosyl(1-2)glycerate for the stabilisation and preservation of biomaterials

Info

Publication number: EP1616005B1
Application number: EP03717817A
Authority: EP
Inventors: Helena Santos; Pedro Miguel Lamosa ANTÓNIO; Carla Jorge; Milton Simoes Da Costa
Original assignee: Instituto de Biologia Experimental e Tecnologia (IBET)
Current assignee: Instituto de Biologia Experimental e Tecnologia (IBET)
Priority date: 2003-04-22
Filing date: 2003-04-22
Publication date: 2009-03-04
Anticipated expiration: 2023-04-22
Also published as: DE60326494D1; EP1616005A1; JP2006514678A; CA2522828A1; WO2004094631A1; ATE424453T1; US20060246028A1; AU2003222537A1

Abstract

The present invention consists in the utilization of glycosyl(1-2)glycosyl(1-2)glyceryls, like mannosyl(1-2)glucosyl(1-2)glycerate, or mixtures thereof alone or as constituents in a suitable formulation as protectors and/or stabilisers of enzymes, proteins, antibodies, DNA or RNA molecules, biological membranes, lipossomes, lipid related substances or other cellular components and biomaterial against general stress.

Description

FIELD OF THE INVENTION

The present invention concerns the use of mannosyl(1-2)glucosyl(1-2)glycerate, in any of its possible stereoisomeric forms, alone or as a constituent of formulations to protect and/or stabilize enzymes or other cellular components and biomaterials against general stress, namely caused by heat, high osmolarity, free-radicals, desiccation, freeze-drying, and repetitive use. Said compounds obey to the general formula depicted in Figure 1.

DESCRIPTION OF THE INVENTION AND STATE OF THE ART

The accumulation of low-molecular mass organic solutes such as, trehalose, polyols or ectoines, is a prerequisite for osmotic adjustment of many mesophilic microorganisms. However, very unusual solutes namely, di-myo-inositol phosphate, di-mannosyl-di-myo-inositvl-phosphate, diglycerol phosphate, mannosylglycerate, and mannosylglyceramide, have been identified in thermophilic and hyperthermophilic microorganisms and the intracellular content of these solutes increases in response to stress conditions, such as high osmolarity or high temperature.
Mannosylglycerate and diglycerol phosphate have been studied to a greater extent and have been shown to protect enzymes and proteins in vitro better than compatible solutes from mesophiies [1-3]. Moreover, the application of compatible solutes from thermophilic or hyperthermophilic organisms, as stabilising agents of biomaterials, has been disclosed in several patent applications [4-6].
We have discovered a novel compatible solute in the thermophilic bacterium Petrotoga miotherma, an organism that grows optimally at 55°C, but is able to grow as high as 65°C. When subjected to salt stress this organism accumulates large amounts (above 1 µmol/mg of protein) of a novel di-sugar compound. After extraction, purification, and full spectroscopic characterization, by Nuclear Magnetic Resonance, we have determined the molecular structure of this, to date, unknown compound as α(1-2)mannopyranosyl-α(1-2)glucopyranosyl-glycerate.
It is interesting to note that the molecular structure of this compatible solute comprises the two moieties (mannosyl and glyceryl) present in mannosylglycerate, a solute widely distributed among thermophiles and hyperthermophiles [7]. In addition, there is a glucosyl moiety linking the mannosyl and glyceryl moieties.
Mannosylglycerate is a well-known biostabiliser of thermophilic origin, whose industrial application is protected under a European patent application [4]. The thermophilic origin of the novel solute combined with the structural resemblance to mannosylglycerate leads us to propose that this novel solute has stabilising properties as good or superior to those already demonstrated for mannosylglycerate. In this respect, it will serve as a stabiliser in various commercial, industrial, medical, pharmaceutical, diagnostic, cosmetic, or academic research applications.
The enhanced protein stability rendered by certain low-molecular mass organic solutes allows enzymes to function under more severe conditions of temperature, pressure, ionic strength, pH, presence of detergents or organic solvents. One of the priorities of modern biotechnology is to obtain stable enzymes or agents that stabilise those enzymes against thermal or chemical denaturation. The ability of some compatible solutes to stabilise enzymes is, therefore, of great importance to modem biotechnology. This point is obviously extended to all proteins that are used or can be used in processes where their stability is an issue, since all proteins, either with or without enzymatic activity, share the same overall basic elements of structure and may be protected against denaturation or inactivation through the same general mechanisms or processes.
II must also be stressed that compatible solutes protect proteins, cell membranes, lipossomes, and cells from the deleterious effects of desiccation, and possess strong moistening properties. The preservation of desiccated or lyophilized cell components and biomaterials has many applications in medicine, pharmaceutical industry, cosmetic industry, food industry, and scientific research. In spite of the great importance of desiccation and freezing in the conservation of biological samples, denaturation of proteins or a decrease of the viable count of cultures inevitably takes place during utilization, and could be prevented or diminished by the use of low molecular mass stabilisers. Also, the stability of nucleic acid molecules, like DNA, or RNA, can be improved by the addition of compatible solutes from hyperthermophiles, as described for ectoines [8], and their use in several applications in medicine, pharmaceutical industry, or scientific research can be envisioned.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 .
Depicts the generic chemical structure of mannosyl(1-2)glucosyl(-2)glycerate in all its possible stereoisomeric forms. The figure is intended to represent both mannosyl and glucosyl moieties either in the α or in the β configuration.

REFERENCES

[1] Ramos, A., N. D. H. Raven, R. J. Sharp, 5. Bartolucci, M. Rossi, R. Cannio, J. Lebbink, J. van der Oost, W. M. de Vos and H. Santos. 1997. Stabilisation of enzymes against thermal stress and freeze-drying by mannosylglycerate. Appl. Environ. Microbiol. 63:4020-4025.
[2] Borges, N., A. Ramos, N. O. H. Raven, R. J. Sharp, and H. Santos. 2002. Comparative study of the thermostabilising properties of mannosylglycerate and other compatible solutes on model enzymes. Extremophiles 6:209-216.
[3] Lamosa, P., A. Burke, R. Peist, R. Huber, M. Liu, G. Silva, C. Rodrigues-Pousada, J. LeGall, C. Maycock, and H. Santos. 2000. Thermostabilisation of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus. Appl. Environ. Microbiol. 66:1974-1979.
[4] Santos, H., A. Ramos, M. S. da Costa. Thermostabilisation, osmoprotection, and protection against desiccation of enzymes, cell components and cells by mannosylglycerate. EP-A-0 816 509 .
[5] Santos, H., P. Lamosa, C. Maycock, and A. Burke, 1999. Thermostabilisation, osmoprotection, and protection against desiccation of enzymes, cell components and cells by di-glycerol-phosphate. EP-A-O 965 268 .
[6] Santos, H., L O. Martins, L. 5. Carreto, and M. 5. da Costa. 1996. Utilização de fosfato de di-manosil-di-mio-inositol e de fosfato de 1,3'- di-mio-inositol na termoestabilisação, osmoprotecção e protecção contra a desidratação de componentes celulares e células. Portuguese patent no. 101813 .
[7] Santos. H., and M. 5. da Costa. 2009. Organic solutes from thermophiles and hyperthermophiles. Methods Enzymol. 334:302-315.
[8] Malin G, Iakobashvili R, and Lapidot A (1999) Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type II restriction endonucleases as a model system, J. Biol. Chem. 274:6920-6929.

Claims

The use of mannosyl(1-2)glucosyl(1-2)glycerate wherein the compound is in any of its possible stereoisomeric forms, alone or as a constituent in a suitable formulation to protect and/or stabilise enzymes or other materials of biological origin against stress caused by heat, high osmolarity, free-radicals, desiccation, freeze-drying, and/or repetitive use.
The use of mannosyl(1-2)glucosyl(1-2)glycerate according to claim 1 wherein the glucosyl and mannosyl residues are in the α or β configurations.
The use of mannosyl(1-2)glucosyl(1-2)glycerate according to claims 1 and 2 wherein the glycerate residue is in the D or L configuration.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 to protect enzymes or other proteins against temperature denaturation induced by purification, transport and/or storage.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 as a protector of the activity of polymerase chain reaction (PCR) enzymes for clinical, biological and industrial purposes during the storage as well as the high-temperature recycling of the enzymes.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 as a stabiliser of enzymes or other proteins during lyophilisation, desiccation or freeze-drying and storage.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 as a stabilising agent during the manufacture, storage and assays using test kit enzymes, for commercial, biological and industrial purposes.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 to stabilise enzymes or other proteins during their routine use for commercial, biological and industrial purposes.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 for the protection or stabilisation of antibodies for commercial, biological, research and industrial purposes.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 for the protection or stabilisation of vaccines of proteic or nonproteic nature for commercial or industrial purposes.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 for the protection of materials of biological origin against stress such as desiccation and lyophilisation of cell membranes, liposomes, liposome-containing cosmetics or lipid related substances.
The use of mannosy(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 for the structural protection or stabilisation of DNA or RNA molecules.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 for the protection, stabilisation or improved performance of DNA or RNA microarrays.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 as an additive to cosmetics to improve moistening properties, stabilise compounds or liposomes or as a suppressor of free radicals.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 to 3 for protection against damage caused by lyophilisation, desiccation, temperature stress, and freezing to microbial cells.
The use of mannosyl(1-2)glucosyl(1-2)glycerate or mixtures thereof according to claims 1 and 4 to 15 wherein the compound is a constituent in a suitable formulation to protect and/or stabilise enzymes or other cellular components and biomaterials in commercial, biological, research, and industrial purposes.